Environ. Sci. Technol. 2006, 40, 2051-2055
Monitoring of Vasculogenesis-Inhibiting Activities in Sewage Effluents by Using Medaka Embryos TAKESHI SUGISE, YASUFUMI HAYASHIDA, TAKAHIRO HANAFUSA, EMI NANJO, AND ICHIRO YAMASHITA* Center for Gene Science, Hiroshima University, Kagamiyama 1-4-2, Higashihiroshima 739-8527, Japan
Environmental samples are known to be contaminated with complex chemicals such as estrogens, polycyclic aromatic hydrocarbons, and retinoids. These contaminants have potentially an adverse impact on survival of aquatic animals, because we found previously that medaka (Oryzias latipes) embryos are defective in the development of blood vessels and bones in the presence of these chemicals. Thus, it is important to test whether sewage effluents contain inhibitory activities against the embryonic development. To examine for such activity, medaka embryos were exposed for 48 h to extracts or freezeevaporated concentrates of effluent samples collected from different municipal sewage treatment plants. We used the transgenic embryos that are hypersensitive to estrogens due to a high-level expression of estrogen receptor for detecting the total (sum of estrogenic and non-estrogenic) vessel-inhibiting activity. The embryos were specifically defective in blood-vessel formation in most effluent samples, showing the activities ranging from 3 to 30 ng of 17βestradiol equiv per liter. Detection limit of 17β-estradiol was 10 ng per liter. For detection of the non-estrogenic vesselinhibiting activity, we treated the transgenic embryos in the presence of an antiestrogen, tamoxifen, or used the wildtype embryos. The non-estrogenic activities were found in some (7 out of 18) effluents, ranging from half to all of the total activities. Our findings for the first time demonstrate the utility of the vascular assay for monitoring sewage effluents.
Introduction In recent years, there is increasing concern about the potential risk in wildlife and humans of exposure to man-made chemicals (1). Unexpected high frequency of intersexes in many fish species living in urban rivers that receive large loads of sewage effluents has been discussed in view of the findings that sewage effluents contain different compounds with estrogenic activity (2-6). Many field and laboratory studies have also shown their effects on induction of yolk protein vitellogenin in male fish (7-10). Natural and synthetic estrogenic steroid hormones, alkylphenol surfactants, plasticizer bisphenol A, pesticides, phytosterols, and others have been identified as potential causative substances (7, 11). * Corresponding author phone: +81-82-424-6271; fax: +81-82424-3498; e-mail:
[email protected]. 10.1021/es051956i CCC: $33.50 Published on Web 02/01/2006
2006 American Chemical Society
However, recent studies indicate that the major estrogenic activity in municipal sewage effluents is derived from steroid hormones secreted from human (12). Other environmental estrogens contribute to minor activities because of their extremely low affinities to estrogen receptor (ER). Because the effluents from industrial, domestic, and agricultural sources are complex mixtures of chemicals, it should be tested whether the effluents contain compounds that affect the embryonic development other than the sexual differentiation in fish. The medaka embryo possesses a unique combination of features that make it particularly well suited for screening of inhibitors against vascular (bloodvessel) formation: transparency, extrauterus development, large yolk veins, and survival in the complete absence of blood circulation. Furthermore, one can easily collect many numbers of fertilized eggs all the year round, and can assay for vessel-inhibiting chemicals in high cost-performance. We reported previously that estrogens, aryl hydrocarbons, and retinoids, all frequently detectable in environmental samples, affect vascular formation in medaka embryos after binding to and activating corresponding nuclear receptors (13-16). We also created transgenic lines expressing a high level of ER under β-actin promoter (15). The transgenic embryos are extremely sensitive to estrogen compared with the wild type and are specifically defective in the formation of yolk veins. The vascular defect is dependent on ER and can be rescued by the addition of an antagonist to estrogen. In this study, we detected blood-vessel-inhibiting activities both estrogenic and non-estrogenic in effluents collected from different sewage treatment plants by using the ERoverexpressing and wild-type medaka embryos. On the basis of these studies, we conclude that the vascular assay may be useful for monitoring vessel-inhibiting activities in effluents.
Materials and Methods Chemicals. 17β-Estradiol (E2), tamoxifen (TAM), all-transretinoic acid (RA), R-naphthoflavone (NF), nonylphenol, bisphenol A, and genistein were obtained from Sigma Chemical Company (Saint Louis, MO). Di-n-butyl phthalate was purchased from Tokyo Kasei Kogyo (Tokyo, Japan). These chemicals were dissolved in dimethyl sulfoxide (DMSO) or ethanol. Extraction and Concentration of Effluent Samples. Sewage effluents (before chlorination) were collected every month from municipal sewage treatment plants and filtered successively through paper filters (Toyo Roshi Kaisha, Japan) and cellulose nitrate membranes with a pore size of 0.45 µm (IWAKI, Japan). Filtrates (2 L) were flowed through a SepPak Plus C18 column (Waters, MA). The cartridge was washed with water, and the bound substances were eluted with methanol. Extracts were dried under a flow of nitrogen gas and dissolved in 0.5 mL of DMSO. Aliquots (e50 µL) of the solution were diluted with 5 mL of Yamamoto’s solution (a saline for medaka) (17) and used in assay. The filtrates (250 mL) were also frozen in dry ice-ethanol and concentrated approximately 5-fold by a vacuum concentrator. Aliquots of the concentrate were mixed with 10× Yamamoto’s solution (0.5 mL), RA (final concentration, 1 nM), and NF (5 µM), adjusted with deionized water to a final volume of 5 mL, and used in assay. Assay for E2 in Effluents by ELISA. Concentrations of E2 in effluents were determined with an ELISA method produced by EnBioTech Laboratories (Tokyo, Japan) after extraction with the C18 cartridge. Samples and standards were analyzed in triplicate. Data are shown as mean. VOL. 40, NO. 6, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Inhibition of yolk-vein development in the transgenic embryos treated with E2. The wild-type and transgenic (Tg) embryos were treated with E2 (1.0 µg/L) at 35 °C for 48 h and photographed in front and side views. Arrows and arrowheads indicate blood clots and yolk veins, respectively. Fish and Assay for Vessel-Inhibiting Activities. We used the d-rR strain (18) of medaka fish, O. latipes, and its transgenic “A”-line homozygous for β-actin-promoter-driven medaka ER cDNA (15). The fish were maintained at 25-26 °C under an artificial photoperiod of 14L:10D, and fed by powdered Tetramin (Tetra, Germany). Eggs were collected within 10-h postfertilization (hpf), rinsed with tap water, and immersed in Yamamoto’s salt solution. Incubation with chemicals and effluent samples started using 10-hpf eggs. At least 30 eggs were used in each experiment. The stock solutions of chemicals were diluted over 1000-fold with Yamamoto’s solution. The solvents were added to the mock-treated eggs as a control. Eggs were incubated at 35 °C for 48 h unless otherwise indicated. Treatments with RA were done under shading. Eggs were inspected for yolk vein under a dissecting microscope and counted for vascular damages (agenesis and degeneration of yolk vein, and blood clotting) (15). Data are presented as a percentage of embryos with the vascular damages in the total embryos used and as an average of two experiments or mean ( standard error of mean (SEM). Differences between the experimental and control groups were analyzed with the student t test. Differences were considered significant at P < 0.05.
FIGURE 2. Vessel-inhibiting activities of E2 and environmental estrogens. (A) The transgenic embryos were incubated in the Yamamoto’s solution containing E2 at the indicated concentrations at 26 °C for 72 h (O) and at 35 °C for 48 h (b) and counted for the vascular damages shown in Figure 1. Data are presented as a percentage of embryos with the vascular damages in the total embryos used. The embryos were also treated with E2 (1.0 µg/L) plus TAM (2.0 mg/L) at 35 °C for 48 h (2). (B) The transgenic embryos were incubated in the Yamamoto’s solution containing E2 at the indicated concentrations, RA (1.0 nM) and NF (5.0 µM), and counted for the vascular damages. Also treated with E2 (100 ng/L) plus TAM (2.0 mg/L) (2). (C) Embryos were incubated in the Yamamoto’s solution containing RA (1.0 nM), NF (5.0 µM), and each of nonylphenol (NP), bisphenol A (Bis), di-n-butyl phthalate (BP) at a concentration of 200 µg/L, and genistein (Gen) at 1.0 mg/L. Closed bar, the transgenic embryos; open bar, the transgenic embryos in the presence of TAM (2.0 mg/L); gray bar, the wild-type embryos. Asterisks indicate statistically significant differences to controls (in the absence of E2 or TAM). **, P < 0.01; ***, P < 0.001.
Results Vessel-Inhibiting Activities of E2 and Environmental Estrogens. We first determined the vessel-inhibiting activitiy of E2 using the wild-type and transgenic embryos. The embryos were incubated at 26 and 35 °C for 72 and 48 h, respectively, at concentrations of E2 ranging from 0.1 to 1.0 µg/L. The wild-type embryos grew normally and developed yolk veins (Figure 1, arrowhead) at all the concentrations tested, showing less than 5% embryos with vascular damages. The transgenic embryos also grew normally in body size at both temperatures but were specifically defective in the formation of yolk veins with blood clots (Figure 1, arrow) on the yolk. The rates of embryos with vascular damages (% embryos) increased in proportion to the increasing concentrations of E2 (Figure 2A), and used as the standard for calculation. Sensitivity to E2 was higher at 35 than 26 °C. The lowest effective concentration of E2 was 0.1 µg/L at 35 2052
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°C. The embryos completely recovered from the vascular inhibition in the presence of an antiestrogen, TAM (Figure 2A), indicating that the vessel-inhibiting activity was estrogenic. Since sewage effluents usually contain ∼10 ng of E2 per liter, we tried to sensitize the vascular assay to ng/L levels of E2 by adding vascular inhibitors such as RA and NF (14, 16). These reagents raised a basal level of the vascular inhibition to approximately 25% but also improved the sensitivity to E2 10-fold (Figure 2B). The lowest effective concentration of E2 was 10 ng/L. Addition of TAM completely abolished the vascular inhibition again. The wild-type embryos showed the vascular inhibition of approximately 20-30% (not significantly different with the basal level for the transgenic embryos) at all the concentrations of E2 tested (data not shown).
TABLE 1: Vessel-Inhibiting Activities in Effluents (in ng/L)a
FIGURE 3. Assay for the vessel-inhibiting activity in effluents. The solid-phase extracts from the effluent samples (A-L) were diluted 100-fold with Yamamoto’s solution and assayed in the transgenic embryos in the absence (closed bar) or the presence (open bar) of TAM (2.0 mg/L). The diluted samples (C and H) were also assayed in the wild-type embryos in the absence of TAM (gray bar). Environmental estrogens such as nonylphenol, bisphenol A, di-n-butyl phthalate, and genistein were examined for the vessel-inhibiting activity in the presence of RA and NF (Figure 2C). Only bisphenol A showed the activity at 200 µg/L, which was estrogenic because no activities were detectable in the presence of TAM or in the wild-type embryos. As an isoflavonoid, genistein is reported to be detectable in urban rivers and pulp mill effluents at relatively high concentrations (10-100 µg/L) (19, 20) and is known as an inhibitor of receptor tyrosine kinases responsible for vessel formation in mammals (21); we also examined for its activity at 1.0 mg/L. Genistein showed the vascular inhibiting activity in medaka embryos, but its activity was assigned to be estrogenic because no activities were detectable in the presence of TAM or in the wild-type embryos. Assay for Vessel-Inhibiting Activities in Effluents. Effluent samples were collected from three different sewage treatment plants in Japan: samples A-C, D-G, and H-L from different plants. The solid-phase extracts were diluted 100-fold and more with Yamamoto’s solution and assayed for the total (estrogenic plus non-estrogenic) vessel-inhibiting activity in the transgenic embryos. The extracts A-C and H-L showed relatively high activities and the extracts D-G low but significant activities: data for the assay with 100fold dilution are presented as closed bars in Figure 3. These extracts showed the activities in a concentration-dependent manner (data not shown). To detect non-estrogenic activities, the extracts were assayed in the presence of the antiestrogen, TAM. The extracts B, C, and H-L showed significant activities (as presented by open bars in Figure 3), indicating that these extracts contain non-estrogenic vessel-inhibiting activities. The extracts C and H were also assayed in the wild-type embryos, resulting in the activities (shown by gray bars in Figure 3) similar to those for the transgenic embryos in the presence of TAM. Table 1 summarizes the vessel-inhibiting activities (E2 equivalents) of effluent samples calculated from the vascular assay and the ELISA assay for E2. The vessel-inhibiting activity for each effluent was divided into the estrogenic and nonestrogenic activity. The latter was calculated from the data in the presence of TAM. The estrogenic activity was calculated by subtracting the non-estrogenic value from the total one in the absence of TAM. We found the following: that major vessel-inhibiting activities were estrogenic in the extracts A and C; that major activities in the extracts B and H-L were non-estrogenic; and that the extracts D-G contained antiestrogenic compounds or other inhibitors (see Discussion). Additional effluents (M-R) were collected from another sewage treatment plant, concentrated approximately 5-fold
effluent
estrogenic
non-estrogenic
E2 (ng/L)b
A B C D E F G H I J K L M N O P Q R
7.9c 5.6c 9.6c 2.1d 2.3d 2.9d 4.4c 3.3d 3.6d ND 2.9 4.2d 7.1c 8.0c 3.2d ND ND ND
2.1 8.9c 4.4c 2.2 NDe ND 2.4 13.8c 13.9c 29.4c 10.3c 7.0c ND ND ND ND ND ND
6.9 5.9 5.7 5.3 4.1 5.3 20 2.6 3.0 2.8 2.5 2.5 5.0 3.4 5.5 5.0 4.2 3.5
a Vessel-inhibiting activities (E2 equivalents) were calculated from values for percentage of embryos with vascular damages and concentration rates of effluents on the basis of the E2 dose-response standards shown in Figures 2A and B. b Measured by ELISA. c Two asterisks ) P < 0.01. d One asterisk ) P < 0.05. e Not detectable.
with a vacuum concentrator, and assayed in the presence of RA and NF. The transgenic and wild-type embryos were used to assay total and non-estrogenic activities, respectively. Data are shown in Table 1. The extracts M, N, and O showed significant activities in the transgenic embryos but no activities in the wild-type embryos, indicating that most of the activities were estrogenic. Although the extracts P, Q, and R contained sufficient amounts of E2 to raise the estrogenic activity, they did not show any vessel-inhibiting activity in both transgenic and wild-type embryos, indicating the presence of abundant inhibitors against E2. The extract O was also considered to contain inhibitors to some extent.
Discussion The purpose of this study was to examine the usefulness of vessel-inhibiting activity as a biomarker to monitor sewage effluents. We used the transgenic medaka embryos rendered hypersensitive to estrogens by overexpressing ER for the detection of estrogenic activity, the environmental contamination of which has become a serious concern in industrial nations. We also used the wild-type embryos for monitoring non-estrogenic vessel-inhibiting activities that may affect embryonic development of aquatic animals. We first assessed the specificity and sensitivity of the assay by measuring responses to E2 and known environmetal estrogens. Inhibition of vessel formation was specifically induced only 2 days after exposure to E2 with the lowest effective concentration of 10 ng/L, equivalent to those detected in urban sewage effluent (22-25). However, the sensitivity was not enough to detect environmental estrogens with extremely low affinities to ER. Only bisphenol A was detectable at a relatively high concentration (200 µg/L). The present assay is convenient and cost-effective but slightly lower in sensitivity than the popular yeast reporter assay with detection limits of E2 ranging from 3 to 20 ng/L (4, 5, 12, 26, 27). Two other transgenic fish, zebrafish (28) and medaka (29), have been developed to detect estrogenic substances. They express luciferase and a green fluorescence protein, respectively, under estrogen-responsive promoters. Detection limits of E2 are reported to be ∼300 pM (∼82 ng/ L) in zebrafish and 630 pM (170 ng/L) in medaka. Although fish models can evaluate total effects of estrogens on living organisms as a sum of uptake, bioconcentration, and metabolism, and thus are useful to monitor sewage effluents, VOL. 40, NO. 6, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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improvements of transgenic fish or assay conditions will be required in order to enhance the sensitivity to estrogens and to monitor sewage effluents continuously without extraction and concentration of effluents. Vessel-inhibiting activity was detectable in most of the effluent samples collected from different sewage treatment plants after extraction with the C18 cartridge or concentration with the vacuum evaporator. The latter method is simple and unrelated to chemical nature of substances and thus may be superior to the former in order to routinely monitor the total estrogenic activity in effluents. Most of the estrogenic activity in the effluents was due to E2, in accordance with the previous results (12), but in two samples (C and N) we detected more estrogenic activities than those calculated from the concentrations of E2. Although it is presently unknown what was responsible for the extra activity, it is reasonable to speculate, besides the possibility for the presence of other estrogens, that the samples contained stimulators for the estrogenic activity such as retinoids and aryl hydrocarbons, both reported to be present in some effluents (30, 31). On the contrary, in several effluent samples (D-G and O-R), we detected lower estrogenic activities than those expected from the concentrations of E2. These inhibitory activities have been reported previously (27). It was shown that fumic substances, present in water samples, are sticky enough to adsorb estrogens and to diminish their activity. Another possibility is the presence of antiestrogenic substances such as TAM in effluent samples. Recently, some antiestrogens were reported to be ubiquitously present in the aquatic environment (32). The non-estrogenic vessel-inhibiting activity was also detectable in some effluent samples (B, C, and H-L). Human urine and fecal water contain several polyphenols including genistein, which are derived from consumption of a plantbased diet and exhibit the inhibitory activity against in vitro and in vivo angiogenesis (21, 33, 34). The phenolic compounds of plant origin may be present in sewage effluents since genistein is frequently detectable in urban rivers (20) at relatively high concentrations (∼100 µg/L). Although genistein played an estrogenic role, as reported previously (8, 35), in the inhibition of vessel formation in medaka, some of the remaining polyphenols may inhibit vessel formation in a non-estrogenic manner. Other candidates for vessel inhibitors are retinoids, aryl hydrocarbons, and pharmaceutical compounds (36), all frequently detectable in the aquatic environment. Identification of major vessel inhibitors in sewage effluents by using medaka embryos may be most important in future studies. However, our finding that RA and NF inhibit vessel formation synergistically with estrogens points out clearly the importance of our bioassay in monitoring the total vessel-inhibiting activity in effluents.
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Acknowledgments This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology.
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Received for review October 2, 2005. Revised manuscript received December 27, 2005. Accepted January 6, 2006. ES051956I
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